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Gene Therapy: Oncolytic Herpes Simplex Virus
Oncolytic virotherapy with herpes simplex virus type 1 (HSV-1) is based on the virus's ability to lyse tumor cells, without infecting and harming normal tissue. HSV-1 can target many tissues naturally, which makes it a promising virotherapy for many types of cancers. However, further manipulation of the HSV vector's genome can target the virus more specifically to certain cancers (7). Why HSV? HSV-1 is an ideal virus for cancer therapy due to the following characteristics (3): #HSV can infect many cell types and tissues, therefore it can target many different kinds of cancer #HSV is cytolytic, meaning it lyses the cells it infects #HSV's genome is large (152 kb) and has many nonessential genes that may allow for integration of therapeutic transgenes #*A number of these nonessential genes are associated with HSV's ability to infect the central nervous system (neurovirulence) #Many drugs exist to control HSV infection preventing viral replication #HSV uses genetic episomes during latency, or rather, does not insert it's genetic information into the host's chromosomes, like a provirus would. The HSV viral genes remain stabilized in the cytoplasm or nucleus (4). Therefore, there will not be a risk for insertional mutagenesis in the host. Cancer Therapy Procedure: Designing HSV Vectors HSV-1 vectors have selectivity for actively dividing cells and therefore cancer cells. This is because there are functional mutations in viral enzymes required for nucleotide metabolism (thymidine kinase (TK), ribonucleotide reductase (RR) and uracil deglycosylase (UNG)). Because the virus cannot replicate without these enzymes, it must infect a suitable host that expresses these enzymes. Cellular enzymes have many similarities to these viral enzymes and are upregulated in actively dividing cells, especially cancer cells. Therefore, HSV vectors selectively infect cancer cells in order to satisfy their replicative needs. First Generation Single-Mutant HSV Vectors (3) Early attempts at creating suitable oncolytic HSV-1 vectors involved mutating or knocking out the specific viral genes for nucleotide metabolizing proteins. This could therefore target the virus to specific cancer cells that may express such enzymes at high levels. The first genetically engineered HSV-1 vector, dlsptk, contained a deletion for the TK gene in its genome. This targeted the virus specifically for brain tumor therapy. Unfortunately, the absence of TK in this virus made it so that antiherpes drugs, acyclovir and ganciclovir, are ineffective at controlling the virus. Normally, these drugs are phospohrylated by thymidine kinase to their precursor active state (5). Additionally, use of the dlsptk vector was discouraged due to neurotoxicity at high viral titers. Since, many other vectors have been created to target different tumors, with varying efficacy and safety. Second Generation Multi-Mutant HSV Vectors (3) Second generation multi-mutant HSV vectors became implemented to decrease the likelihood of a reversion in the virus to it's infectious state. This was done by introducing more than one mutation into the viral vector. One such vector was G207, which had both copies of it's y34.5 gene and the ICP6 gene inactivated by insertion of the LacZ gene from E. coli. This vector was also initially targeted for brain tumor therapy, but studies have revealed that the viral vector works effectively against many solid tumors of different tissues, including breast, gallbladder, colon, melanoma, gastric head, neck, ovarian, pancreatic and prostate cancers. Studies revealed that at high doses, these viruses did not cause any adverse effects in mice or Aotus monkeys. Not only does introduction of these two mutations increase safety of the viral vector, but it also allows one to track the virus within the host. The LacZ gene can serve as an indicator by PCR analysis for infection of certain tissues. Third Generation Multi-Mutant HSV Vectors (6) Third generation HSV vectors evolved to increase MHC I expression on infected cells, which is normally reduced or inhibited by wild type HSV. Specifically, G207 was converted to G47Δ by delecting the alpha-47 gene in the viral vector. This gene normally inhibits the transporter associated with antigen presentation in infected host cells. When this gene is deleted, down regulation of MHC I expression does not occur and the hosts immune system can further attack infected tumor cells. In additon, HSV vectors can include immunostimulatory genes, such as interleukin 4, to enhance antitumor activity. Potential Adverse Effects One must give careful thought into designing an appropriate HSV vector for the type of cancer intended for target. Some of the potential adverse effects that can occur are: *Viral genome reversion to wild type: If the virus reverts to it's wild type phenotype, the virus will be able to infect healthy cells as well as the tumor cells. To prevent this from occuring, multiple genes in the virus should be mutated, such as those discussed in Second Generation Vectors. *Neurotoxigenicity: If the virus is capable of infecting cells in the central nervous system, high viral titers required to kill and prevent tumor growth may also cause neurotoxicity. *Reduced HSV activity in those infected with HSV prior to treatment: Those who have been exposed to HSV may have high antibody titers against the virus, reducing efficacy of the treatment for cancer. A Specific Case: Treatment of Breast Cancer HSV-1 has many applications within oncology, including breast cancer. Breast cancer is the second leading cause of cancer-related death to women in the United States and also recurs in about 30% of patients with early-stage disease. Therefore, treatment of breast cancer with HSV-1 would be ideal to more effectively inhibit recurrence of breast cancer by targetting cancer stem cells that are the primary cause of recurring and metastatic breast cancer. Li et al. tested whether HSV G47Δ was effective at inbhiting breast tumor growth by targeting breast cancer stem-like cells with promising results.http://www.nature.com/nbt/journal/v30/n7/full/nbt.2287.html Description of G47Δ *Deletion of both copies of the y34.5 gene: prevention of neurovirulence *LacZ insertion into IPC6 gene: viral ribonucleotide reductase is deleted which is necessary for viral replication in non-dividing cells. This targets the virus to actively dividing tumor cells. *Deletion of IPC47 gene and US11 promoter: enhancement of immunogenicity Methods and Results Li et al. grew SK-BR-3 and primary breast cancer cell lines in vitro, generating mammospheres which had cancer stem cell properties. These mammospheres were implanted subcutaneously in mice and were determined to be highly tumorigenic. Infection of stem-cell like cancer cells with G47Δ in vitro revealed high toxicity to the stem cells.'' In vivo'' infection of mammospheres, determined by X-gal staining for LacZ, revealed significant reduction in tumor growth compared to control. The G47Δ infected tumors also had fewer stem-like cells, identified by flow cytometry staining. Find the full articlehere. References #Enhancement of systemic tumor immunity for squamous cell carcinoma cells by an oncolytic herpes simplex virus. http://www.nature.com/cgt/journal/v20/n9/pdf/cgt201345a.pdf #Oncolytic herpes simplex virus engineering and preparation. http://www.ncbi.nlm.nih.gov/pubmed/21948465 #Oncolytic herpes simplex virus vectors for cancer virotherapy. http://www.nature.com/cgt/journal/v9/n12/full/7700537a.html #Wikipedia: Virus Latency. http://en.wikipedia.org/wiki/Virus_latency #Wikipedia: Aciclovir. http://en.wikipedia.org/wiki/Aciclovir #"Armed" oncolytic herpes simplex virus for brain tumor therapy. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634086/ #Oncolytic virotherapy. http://www.nature.com/nbt/journal/v30/n7/full/nbt.2287.html